As used herein, certain citations to references are indicated as numerals or alphanumerical symbols in parentheticals or as superscripts, and are further described in the “References Cited” listing contained herein.
Citrate is a primary metabolite that is ubiquitously used as a source of carbon and energy by most living organisms. While there has been extensive research conducted into citrate transport across membranes (A21), there has been a relative dearth of research into membrane protein systems that can transport complexed citrate (A2). Currently, there is predicted to be over 90 members in the so-called CitMHS family of secondary transporters (A25). Members of this superfamily are found in Gram-positive bacteria and are predicted in Gram-negative bacteria. It is believed that these organisms take up complexed citrate, because it is predominantly available as such in their environment or to allow access to critical metal ions such as iron. To date, the only functionally characterized systems for metal-citrate transport in this family are those of Bacillus subtilis (A19), Streptococcus mutans (A20), and most recently Enterococcus faecalis (A4). These members of the CitMHS family transport metal-citrate complexes in symport with one H+ per M2+-citrate.
Lolkema et at demonstrated that CitM from B. subtilis transported citrate in complex with Mg2+, Ni2+, Mn2+, Co2+ and Zn2+ but not in complex with Ca2+, Ba2+ and Sr2+. CitH, also from B. subtilis, transported citrate and iso-citrate complexed to Ca2+, Ba2+ and Sr2+ but not Mg2+, Ni2+, Mn2+, Co2+ and Zn2+ (A19, A31). The group of metal ions transported by CitM includes the smaller cations, with a Pauling radius of less than ˜0.80 Å. The ions transported by CitH of B. subtilis have radii larger than 0.98 Å. Neither transporter was shown to transport free citrate or metal complexes of other tri-carboxylates (or similar dicarboxylates) such as cis-aconitate and tricarballylate. More recently, Cvitkovitch et al. functionally characterized the CitM homolog from Streptococcus mutans (A20). Citrate complexed to Fe3+ and Mn2+ was transported with S. mutans, however Ca2+, Mg2+ and Ni2+ were not. The CitH transporter of Enterococcus faecalis was characterized in 2006 (A4). High amino acid (AA) sequence homology to that of S. mutans led researchers to believe it could be using a CitMHS transporter to access iron. In fact this was shown not to be the case. The system was shown to be a CitM (B. subtilis) functional homolog, with larger ionic radii metals such as Ca2+, Sr2+, Cd2+ and Pb2+ involved in transport but not Fe2+ or Fe3+. This unpredictability clearly demonstrates the limited understanding of these systems.
Pathogenic bacteria are an important health concern worldwide. Some examples of pathogenic bacteria include Bacillus anthracis, Mycobacterium tuberculosis, Corynebacterium diphtheriae, Neisseria meningitis, and Neisseria gonorrhoeae. Pathogenic bacteria are also a health concern for animals. Because pathogenic bacteria cause infectious diseases, some of which cause millions of deaths worldwide each year, it is important that diagnostic tools and treatments that target pathogenic bacteria continue to be developed. A better understanding of the mechanisms by which pathogenic bacteria uptake nutrients and cause infection is needed to develop better prevention and treatement regimes against these bacteria.
The genome of Streptomyces coelicolor was sequenced in 2002 (A14). This effort identified an unprecedented number of genes encoding membrane-spanning transporters and gene sets that would encode enzymes for utilizing complex nutrients. The transporters on the S. coelicolor phylogenetic branch were found to share only between 35-45% amino acid (AA) sequence homology with those transporters investigated to date compared to 60-83% AA homology between the B. subtilis, E. faecalis, and S. mutans transporters. However, since 2002, there has been no reports on the identification and functional characterization of a metal-citrate transport of S. coelicolor. Thus, prior to the present invention, there was a need for such information, particularly since this information could lead to diagnostic tests and treatments for pathogenic bacteria.
The present invention is directed to the deficiencies in the art.